CN1526014A - Paramyxovirus vector for introducing foreign genes into skeletal muscle - Google Patents

Abstract

Whether recombinant Sendai virus (SeV) vector can be used for transporting genes into skeletal muscle was examined using LacZ reporter gene and insulin-like growth factor gene. As a result, transgene expression continued at longest for 1 month after the injection. Compared with control, the transduction of the insulin-like growth factor gene caused a significant increase in regenerated fibers and splitting myofibers, i.e., an index of hypertrophy. Furthermore, the total number or myofibers increased by the gene. Thus, Paramyxovirus vectors, including Sendai virus, were shown to achieve a high-level expression of transgenes in skeletal muscle; and the high potential of the transduction of an insulin-like growth factor gene using a Paramyxovirus vector in treating neuromuscular disorders was indicated.

Description

Paramyxovirus vector for introducing foreign genes into skeletal muscle

technical field

The present invention relates to a paramyxovirus vector for introducing foreign gene into skeletal muscle.

Background technique

The subfamily Paramyxovirinae includes enveloped viruses of the three genera Rhabdoviridae, Paramyxoviridae and Filoviridae, which have non-segmented (-) stranded RNA genomes, The genome functions as a template for the synthesis of mRNA and anti-strand genes. Sendai virus (SeV) belongs to the genus Paramyxovirus, and it has not been considered pathogenic to humans since the initial research of virology (Virus Experiment Report, Miyuki Nagai, Akira Ishihama, 1995.4). SeV has a strict cytoplasmic life cycle in mammalian cells, and the introduced RNA does not interact with host cell chromosomes and remains in the cytoplasm. Therefore, SeV can be safely used in human gene therapy. The full-length genomic nucleotide sequence of the SeVZ strain used here was determined in 1986 by Shibuta et al. Also, infectious virus was successfully recovered by transforming SeV cDNA, which made possible genetic manipulation (Conzelmann, K.K.Annu.Genet., 1998, 32, 123-162; Y.Nagai, A.KatoMicrobiol.Immunol., 1999 , 43, 613-624). Furthermore, various exogenous genes with additional transcription units were inserted into appropriate positions in the genome and expressed at high levels in SeV (Yu, D., T. Shioda, A. Kato, M.K. Hasan, Y, Sakai and Y. Nagai, Genes Cells., 1997, 2, 457-466).

Insulin-like growth factor (IGF-I) plays an important role in the development, maintenance and regeneration of skeletal muscle. The effect of IGF-I on myogenic cells includes promoting myoblast replication, myogenic differentiation and causing myotube hypertrophy (Annu.Rev.Physiol., 1991,53,201-216; Endocr.Rev., 1996,17,481 -516; Cell., 1993, 75, 59-72; Genes Dev., 1993, 7, 2609-2617). Muscle regeneration involves proliferation, fusion with myotubes, and reinnervation of muscle precursor cells. IGF-I produced in satellite cells acts as a potent stimulator of proliferation and differentiation of muscle precursor cells (Acta Physiol Scand, 1999, 167, 301-305). The introduction of IGF-I gene into skeletal muscle has been applied in the following aspects: treatment of denervated skeletal muscle atrophy with non-viral technology and treatment of age-related skeletal muscle decline with AAV vector (Proc.Natl.Acad.Sci.USA. , 1998, 95, 15603-15607).

invention disclosure

The object of the present invention is to provide a paramyxovirus vector capable of efficiently introducing foreign genes into skeletal muscle cells and its application. Specifically, the present invention provides a paramyxovirus vector capable of introducing exogenous genes into skeletal muscle, a composition containing the vector for introducing exogenous genes into skeletal muscle, and a A method for introducing a source gene into skeletal muscle. In a preferred embodiment of the present invention, a paramyxovirus vector inserted with a gene encoding insulin-like growth factor and its use in muscle fiber formation are provided.

An ideal site for delivery and expression of exogenous genes encoding therapeutic proteins for the treatment of systemic diseases and neuromuscular disorders is skeletal muscle. The present inventors investigated whether a foreign gene can be introduced into skeletal muscle using a recombinant Sendai virus (SeV) vector containing a LacZ reporter gene and a human insulin-like growth factor (hIGF-I) gene.

Seven days after intramuscular injection of LacA/SeV, numerous X-gal-labeled myofibers were found in bupivacaine-treated/untreated animals, and transgene expression persisted for up to a month after injection. The main protein species detected in the culture supernatant of L6 cells was recombinant hIGF-I derived from IGF-I/SeV. Therefore, the induced L6 cells underwent morphological changes such as multinuclear remodeling and hypertrophy. When IGF-I/SeV was introduced into muscle, compared with the control group treated with LacA/SeV, it was found that regenerated muscle fibers and split muscle fibers as hypertrophy indicators increased significantly, and the total number of muscle fibers also increased. These results indicate that paramyxovirus vectors carrying Sendai virus achieve high levels of transgene expression in skeletal muscle, and therefore it is likely that the use of paramyxovirus vectors to introduce the IGF-I gene is effective in the treatment of neuromuscular disorders.

The present invention relates to: a paramyxovirus vector for introducing exogenous gene into skeletal muscle, a method for introducing exogenous gene into skeletal muscle by using the vector, a paramyxovirus vector inserted with insulin-like growth factor as exogenous gene and its Applications in myofiber formation. Specifically, the present invention provides:

(1) A method for introducing a foreign gene into skeletal muscle, the method comprising administering a paramyxovirus vector inserted with a foreign gene to skeletal muscle.

(2) The method according to item (1), wherein the paramyxovirus is Sendai virus.

(3) The method according to item (1) or (2), wherein the foreign gene is a therapeutic gene.

(4) The method according to item (1) or (2), wherein the foreign gene is a gene encoding insulin-like growth factor.

(5) A paramyxovirus vector inserted with a foreign gene for introducing the foreign gene into skeletal muscle.

(6) The vector according to item (5), wherein the paramyxovirus is Sendai virus.

(7) The vector according to item (5) or (6), wherein the foreign gene is a therapeutic gene.

(8) The vector according to item (5) or (6), wherein the foreign gene is a gene encoding insulin-like growth factor.

(9) A composition for introducing a foreign gene into skeletal muscle, the composition comprising a paramyxovirus vector inserted with the foreign gene.

(10) The composition according to item (9), wherein the paramyxovirus is Sendai virus.

(11) The vector according to item (9) or (10), wherein the foreign gene is a therapeutic gene.

(12) The vector according to item (9) or (10), wherein the foreign gene is a gene encoding insulin-like growth factor.

(13) The composition according to item (12), which is used for increasing regenerated muscle fibers and/or split muscle fibers in mammals.

(14) The composition according to item (12), which is used for the treatment of neuromuscular disorders.

In the present invention, the term "paramyxovirus vector" refers to a vector derived from paramyxovirus and capable of introducing genes into host cells. The paramyxovirus vector of the present invention may be a ribonucleoprotein (RNP) or an infectious virus particle. Here, "infectivity" refers to the ability of a recombinant paramyxovirus vector to introduce genes inside the vector into cells to which the vector adheres by maintaining its cell adhesion and membrane fusion capabilities. In a preferred embodiment, the paramyxovirus vector of the present invention integrates expressible foreign genes by means of genetic engineering. The paramyxovirus vector of the present invention may have replication ability, or may be a defective vector without replication ability. As used herein, "replication competence" refers to the ability of a virus to replicate and produce infectious virus particles in a host cell infected with the viral vector.

In this specification, "recombinant" paramyxovirus vector refers to a paramyxovirus vector constructed by genetic manipulation or its amplified product. For example, recombinant paramyxovirus vectors can be prepared by reconstitution of recombinant paramyxovirus cDNA (Y. Nagai, A. Kato, Microbiol. Immunol., 1999, 43, 613-624).

In the present invention, "paramyxovirus" refers to a virus belonging to the family Paramyxoviridae or a derivative thereof. Paramyxoviruses applicable to the present invention include: Sendai virus, Newcastle disease virus, mumps virus, measles virus, respiratory syncytial virus (RSV), rinderpest virus, warm virus (distemper virus), monkey Parainfluenza virus (SV5) and types I, II and III of human parainfluenza virus, etc. The virus of the present invention is preferably a virus of the genus Paramyxovirus or a derivative thereof.

Paramyxoviruses applicable to the present invention include: type I parainfluenza viruses such as Sendai virus and human HA2, type II parainfluenza viruses such as monkey SV5, SV41 and human CA virus, type III parainfluenza viruses such as bovine SF and human HA1, etc. Influenza virus, type IV parainfluenza virus (including subtype A and subtype B), mumps virus, Newcastle disease virus and many other viruses of the genus Paramyxovirus, the paramyxovirus of the present invention is most preferably Sendai virus. These viruses can be natural strains, mutant strains, laboratory passage strains and artificially constructed strains, etc. Incomplete viruses such as DI particles (J. Virol., 1994, 68, 8413-8417), synthetic oligonucleotides, etc. can also be used as materials for preparing the virus vector of the present invention.

Genes encoding paramyxovirus proteins include the NP, P, M, F, HN, and L genes. Here, "NP, P, M, F, HN and L genes" refer to genes encoding nucleocapsid protein, phosphoprotein, matrix protein, fusion protein, hemagglutinin-neuraminidase protein and macromolecular protein, respectively. The genes of each virus in the Paramyxoviridae subfamily are generally indicated below, and the NP gene is also often described as the "N gene".

Paramyxovirus NP P/C/V M F HN - L

Rublavirus NP P/V M F HN (SH) L

Morbillivirus NP P/C/V M F H - L

For example, in the nucleotide sequence database, the entry numbers of Sendai virus classified as Respirovirus in the family Paramyxoviridae are M29343, M30202, M30203, M30204, M51331, M55565, M69046 and X17218 for the NP gene; M30202、M30203、M30204、M55565、M69046、X00583、X17007和X17008；M基因的是D11446、K02742、M30202、M30203、M30204、M69046、U31956、X00584和X53056；F基因的是D00152、D11446、D17334、D17335、 M30202、M30203、M30204、M69046、X00152和X02131；HN基因的是D26475、M12397、M30202、M30203、M30204、M69046、X00586、X02808和X56131；L基因的是D00053、M30202、M30203、M30204、M69040、X00587和X58886.

In the present invention, the term "gene" refers to genetic material, including nucleic acids such as RNA and DNA. Genes may or may not encode proteins, and may encode functional RNAs such as ribozymes or antisense RNAs. A gene can be a natural sequence or an artificially designed sequence. Furthermore, in the present invention, the term "DNA" includes single-stranded DNA and double-stranded DNA.

In the present invention, the term "skeletal muscle" refers to striated muscle, and includes cardiac muscle in the present specification.

The invention provides the application of the paramyxovirus vector in introducing gene into skeletal muscle. The present inventors found that Sendai virus of the genus Paramyxovirus can efficiently transfer genes into skeletal muscle, which is an important target for the treatment of systemic diseases and neuromuscular disorders. Therefore, the vector of the present invention is suitable for gene therapy of the above-mentioned diseases.

Furthermore, the present inventors revealed that the expression of genes introduced into skeletal muscle using recombinant Sendai virus vectors can be sustained for 1 month. This suggests that gene therapy targeting skeletal muscle with recombinant Sendai virus vectors can achieve sustained efficacy.

In terms of safety, since the paramyxovirus vector is not pathogenic to humans, it means that it is preferably used in clinical trials of human gene therapy. First of all, in the expression of exogenous genes caused by plasmid DNA, the main obstacle to efficient gene expression is that the introduced DNA must be transported into the nucleus, or its nuclear membrane must disappear. However, in cases such as Sendai virus, the expression of foreign genes is driven by cellular tubulin in the cytoplasm and its own RNA polymerase (L protein) as the viral genome replicates. This indicates that Sendai virus does not interact with host chromosomes, and it is generally believed that there will be no safety issues such as cell canceration caused by chromosomal abnormalities. Second, Sendai virus is known to be pathogenic in rodents and can cause pneumonia, but it is not pathogenic in humans. One piece of evidence is that intranasal administration of wild-type Sendai virus to nonhuman primates does not cause serious harm (Hurwitz J.L. et al., Vaccine, 1997, 15, 533-540). These characteristics suggest that the Sendai virus vector can be used in human therapy, and it is also expected to become an option for gene therapy of skeletal muscle.

Therefore, the present invention finds that paramyxovirus vectors are effective in introducing genes into skeletal muscle, and this discovery may bring great progress to gene therapy, especially gene therapy targeting skeletal muscle.

In the present invention, the recombinant paramyxovirus vector used for gene introduction into skeletal muscle is not limited to any particular kind. For example, suitable paramyxovirus vectors include those capable of replication and self-propagation. Typically, the genome of wild-type paramyxoviruses contains a short 3′ leader region followed by six genes encoding N (nucleocapsid), P (phosphorus), M (matrix), F (fusion), HN (blood lectin-neuraminidase) and L (large) protein with a short 5' tail region at the other end. The self-replicating vector of the present invention can be obtained by designing a genome having the same structure as above. In addition, a vector expressing a foreign gene can be obtained by inserting a foreign gene into the genome of the above-mentioned vector. In the paramyxovirus vector of the present invention, the arrangement of viral genes may be different from that of the wild-type virus.

The paramyxovirus vectors used in the present invention may lack certain genes contained in the wild-type virus. For example, the proteins encoded by the NP, P/C, and L genes are generally considered to be essential when reconstructing the Sendai virus vector, but the viral vector of the present invention does not necessarily contain the genes encoding the above proteins as its constituents. For example, an expression vector carrying a gene encoding the above protein can be co-transfected into a host cell with an expression vector encoding the vector genome, thereby reconstituting a viral vector. Alternatively, the expression vector encoding the vector genome is introduced into a host cell carrying the gene encoding the above-mentioned protein, and the above-mentioned protein is provided by the host cell, thereby reconstituting the viral vector. The amino acid sequences of these proteins can be different from those from the starting virus, as long as they have equivalent or stronger activity compared with the natural type during nucleic acid introduction, they can be mutated or replaced by homologous genes from another virus .

It is generally believed that the proteins encoded by the M, F and HN genes are necessary for cell-cell propagation of paramyxoviral vectors. However, these proteins are not required when the viral vector is in the RNP form. If the genome in the RNP contains M, F, and HN genes, when it is introduced into a host cell, the products of these genes are produced and infectious virus particles are produced.

RNP can be combined with transfection reagents such as lipofectamine and polycationic liposomes to form a complex and then introduced into cells. Specifically, various transfection reagents can be used, for example, DOTMA (Boehringer), Superfect (QIAGEN #301305), DOTAP, DOPE, DOSPER (Boehringer #1811169) and the like. Chloroquine can be added to prevent degradation in endosomes (Calos, M.P., Proc. Natl. Acad. Sci. USA, 1983, 80, 3015). In the case of a replicating virus, the produced virus can be amplified or passaged by reinfecting cultured cells, chicken eggs, or animals (such as mammals such as mice).

Conversely, paramyxovirus vectors lacking M, F and/or HN genes can also be used as paramyxovirus vectors of the present invention. These vectors can be reconstituted, for example, by exogenously supplying the missing gene product. The viral vector prepared in this way can still adhere to the host cells and induce cell fusion like the wild-type virus. But progeny virus particles do not have the same infectivity as the original virus because the vector genome introduced into the cells lacks one of the above-mentioned genes. Therefore, these vectors can be used as safe viral vectors capable of gene transfer only once. For example, the gene deleted in the genome can be the F and/or HN gene. The viral vector can be reconstructed as follows: co-transfect the expression plasmid of the recombinant paramyxoviral vector genome that lacks the F gene, the expression vector encoding the F protein, and the expression vectors encoding the NP, P/C, and L proteins into host cells (International Application Nos. PCT/JP00/03194 and PCT/JP00/03195); alternatively, a host cell in which the F gene is integrated in the chromosome can be used. When these protein groups are provided exogenously, their amino acid sequences may be different from those of the wild-type virus; and, as long as the activity in nucleic acid introduction is equivalent to or stronger than that of the natural type, they may be mutated or replaced by other A viral homologous gene substitution.

Vectors can be prepared that contain, among the envelope proteins, a different protein than the viral envelope protein encoded by the vector genome. The protein is not limited and may include envelope proteins of other viruses, such as the G protein of vesicular stomatitis virus (VSV) (VSV-G). The paramyxovirus vectors of the present invention include pseudotyped virus vectors such as VSV-G, which have envelope proteins derived from viruses other than genome-encoded viruses.

The paramyxovirus vector of the present invention also contains the following proteins on the surface of the viral envelope, for example: proteins such as adhesion factors, ligands and receptors that can adhere to specific cells, or chimeric proteins (which contain The above-mentioned proteins include viral envelope-derived polypeptides inside the virus). This enables the generation of vectors targeted to specific tissues. These proteins can be encoded by the viral genome itself, or can be expressed by genes outside the viral genome (for example, genes of other expression vectors or host cell chromosomes) when the viral vector is reconstituted.

Viral genes contained in the viral vectors of the present invention may be altered, for example, to reduce antigenicity or to increase RNA transcription efficiency or replication efficiency. Specifically, at least one of the replication factor NP, P/C and L genes can be altered to enhance transcription or replication function. The HN protein, one of the structural proteins, has hemagglutinin activity and neuraminidase activity. If the former activity can be weakened, the stability of the virus in blood may be improved; changing the latter activity may regulate the infectivity. The fusion ability of membrane-fusogenic liposomes can be modulated by altering the F protein, which is involved in membrane fusion. For example, paramyxoviruses with weakened antigen-presenting ability are prepared by analyzing the antigen-presenting epitopes of F protein or HN protein that may become cell surface antigen molecules.

The viral vector of the present invention can encode foreign genes in genomic RNA. A recombinant paramyxovirus vector carrying a foreign gene can be prepared by inserting the foreign gene into the genome of the paramyxovirus vector. The exogenous gene may be a gene that needs to be expressed in the target skeletal muscle. The foreign gene may be a gene encoding a natural protein, or a gene encoding a protein obtained by modifying a natural protein by deletion, substitution or insertion, as long as the modified protein has an equivalent function to the natural protein. For example, for the purposes of gene therapy and the like, a therapeutic gene for a target disease can be inserted into the DNA encoding the viral vector. In the example of introducing a foreign gene into the Sendai virus vector DNA, it is preferable to insert a sequence comprising multiples of 6 nucleotides between the transcription termination sequence (E) and the transcription initiation sequence (S) (Journal of Virology, 1993, 67(8), 4822-4830). Foreign genes can be inserted upstream and/or downstream of each gene (NP, P, M, F, HN and L genes) of the virus. In order not to interfere with the expression of upstream and downstream genes, the E-I-S sequence (transcription termination sequence-intervening sequence-transcription initiation sequence) or a part thereof can be appropriately inserted upstream or downstream of the foreign gene, or the foreign gene can be inserted by means of IRES.

The expression level of the inserted foreign gene can be regulated by the type of transcription initiation sequence linked upstream of the gene, and can also be regulated by the position of the insertion point and the base sequence before and after the gene. For example, in Sendai virus, the closer the insertion point is to the 3'-end of the negative-strand RNA of the viral genome (the closer to the NP gene in the gene arrangement of the wild-type viral genome), the higher the expression level of the inserted gene. In order to obtain high-level expression of the foreign gene, it is preferably inserted into the upstream region of the negative strand genome, such as the upstream of the NP gene (3' flanking sequence on the negative strand), or between the NP and P genes. Conversely, the closer the insertion point is to the 5'-end of the minus-strand RNA (the closer to the L gene in the gene arrangement of the wild-type viral genome), the lower the expression level of the inserted gene. In order to reduce the expression level of the foreign gene, it can be inserted into the 5' most terminal position on the negative strand, that is, the downstream of the L gene of the wild-type viral genome (the 5' flanking region of the L gene on the negative strand) or the upstream of the L gene ( 3' flanking region of the L gene on the negative strand). In this way, the insertion position of the foreign gene can be appropriately adjusted to obtain the desired level of gene expression, or to optimize the combination of the insert and its surrounding genes encoding viral proteins. In order to facilitate the insertion of foreign genes, a cloning site can be designed at the insertion position. For example, the cloning site can be a recognition sequence for a restriction enzyme. In the vector DNA encoding the genome, restriction sites can be inserted into foreign genes. The cloning site may be a multiple cloning site comprising multiple recognition sequences for restriction enzymes. In addition, the vector of the present invention may also have other foreign genes at positions other than the above insertion positions.

A recombinant Sendai virus with a foreign gene can be constructed as follows, for example, according to the methods described in Kato A. et al., EMBO J., 1997, 16: 578-587, and Yu D. et al., GenesCells, 1997, 2: 457-466 carrier.

First, a DNA sample containing the cDNA nucleotide sequence of a desired foreign gene is prepared. Preferably the DNA sample is at a concentration of 25 ng/µl or higher and can be confirmed as a single plasmid by electrophoresis. The following is an example of insertion of a foreign gene into the NotI site of viral genomic DNA. If the target cDNA nucleotide sequence contains a NotI recognition site, it is preferable to modify the nucleotide sequence by site-directed mutagenesis in advance to remove the site, but not to change the encoded amino acid sequence. The desired DNA fragment is recovered from the DNA sample by PCR amplification. In order to obtain an amplified fragment with NotI sites at both ends, and add the Sendai virus transcription termination sequence (E), intervening sequence (I) and transcription initiation sequence (S) (EIS sequence) to one of the ends One copy, a forward DNA sequence and a reverse DNA sequence (antisense strand) were prepared as a primer pair comprising a NotI restriction enzyme recognition sequence, a transcription termination sequence (E), an intervening sequence (I) and a transcription initiation sequence. The original sequence (S), and the partial sequence of the target gene.

For example, the forward synthetic DNA sequence contains any two or more nucleotides (preferably 4 nucleotides, excluding sequences from the NotI recognition site such as GCG and GCC) at the 5'-end, more preferably ACTT ) to ensure digestion with NotI. At the 3'-end of this sequence a NotI recognition sequence GCGGCCGC was added. In addition, arbitrary 9 nucleotides or a multiple of 9 plus 6 nucleotides are added as a spacer sequence at the 3'-terminus. Further, a sequence of about 25 nucleotides of ORF starting from and including ATG, the initiation codon of the desired cDNA, is added to the 3'-terminus. Preferably, the 3'-end of the forward synthetic oligo DNA is selected about 25 nucleotides from the desired cDNA such that the last nucleotide is a G or C.

The reverse synthetic DNA sequence contains any two or more nucleotides (preferably 4 nucleotides, excluding sequences from NotI recognition sites such as GCG and GCC, more preferably ACTT) at the 5'-terminus. At the 3'-end of this sequence a NotI recognition sequence GCGGCCGC was added. In addition, an oligo DNA spacer sequence was inserted at the 3'-end to adjust the length of the primer. The length of oligoDNA is designed according to the following method: comprise NotI recognition sequence GCGGCCGC, the complementary sequence of cDNA and the EIS sequence that is derived from Sendai virus genome described later, the multiple of 6 (so-called " rule of 6 " of nucleotide total number; Kolakofski D . et al., J. Virol., 1998, 72, 891-899). In addition, at the 3'-end of the insert fragment, the complementary sequence of Sendai virus S sequence (preferably 5'-CTTTCACCCT-3'), the complementary sequence of I sequence (preferably 5'-AAG-3'), the complementary sequence of E sequence ( 5'-TTTTTCTTACTACGG-3') is preferred. Further, the complementary sequence of the desired cDNA is added to the 3'-terminus so that the last nucleotide is G or C, and the last nucleotide is located about 25 nucleotides upstream of the stop codon of the cDNA. Use this as the 3'-end of the reverse synthetic oligo DNA.

PCR can be performed by a conventional method using ExTaq polymerase (TaKaRa). Preferably using Vent polymerase (NEB), the amplified fragment of interest is digested with NotI, and then inserted into the NotI site of the plasmid vector pBluescript. The nucleotide sequence of the obtained PCR product was detected by an automatic DNA sequencer, and the plasmid with the correct sequence was selected. The insert was excised from this plasmid by NotI digestion and cloned into the NotI site of a plasmid containing the paramyxovirus genomic cDNA. Alternatively, recombinant Sendai virus cDNA can also be obtained by directly inserting the PCR product into the NotI site without using the pBluescript vector.

The DNA encoding the viral genome is ligated with an appropriate transcription promoter to construct a vector DNA. The obtained vector DNA is transcribed in vitro or in cells, and the RNP is reconstituted in the presence of viral L, P and NP proteins, so that a viral vector containing the RNP can be produced. Reconstruction of viruses from viral vector DNA can be carried out according to known methods (WO97/16539; WO97/16538; Durbin A.P. et al., Virol., 1997, 235, 323-332; Whelan S.P. et al., Proc. Natl. Acad. Sci. USA, 1995, 92, 8388-8392; Schnell M.J. et al., EMBO J., 1994, 13, 4195-4203; Radecke F. et al., EMBO J., 1995, 14, 5773-5784; Lawson N.D. et al., Proc. Natl. Acad .Sci.USA, 1995,92,4477-4481; Garcin D. et al., EMBO J., 1995,14,6087-6094; Kato A. et al., Genes Cells, 1996,1,569-579; Baron M.D. and Barrett T., J. Virol., 1997, 71, 1265-1271; Bridgen A. and Elliott R.M., Proc. Natl. Acad. Sci. USA, 1996, 93, 15400-15404). These methods enable the reconstruction of paramyxovirus vectors from DNA, including parainfluenza, vesicular stomatitis, rabies, measles, rinderpest, and Sendai virus vectors. Infectious virus particles cannot be formed when the F, HN and/or M genes are deleted from the viral vector DNA. However, it is possible to introduce these deleted genes and/or genes encoding other viral envelope proteins from another virus into the host cell and express them so that infectious virus particles can be produced.

The method for introducing vector DNA into cells includes: (1) preparing a DNA pellet capable of being incorporated into desired cells, (2) preparing a complex containing positively charged DNA, which is suitable for incorporating into desired cells and has a lower cellularity. Toxic activity, and (3) use of electrical pulses to open a transient pore in the cytoplasmic membrane of the desired cell, just large enough to allow DNA to pass through.

Various transfection reagents such as DOTMA (Boehringer), Superfect (QIAGEN #301305), DOTAP, DOPE and DOSPER (Boehringer #1811169) can be used in method (2). In method (1), calcium phosphate can be used for transfection. In this method, DNA is taken up by cells in the form of phagocytic vesicles, but DNA is known to be taken up in the nucleus in sufficient quantities (Graham FL and VanDer Eb J., Virology., 1973, 52, 456; Wigler M . and Silverstein S., Cell, 1977, 11, 223). Chen and Okayama studied the optimal conditions for the introduction technology, and they reported that: (1) the incubation conditions of cells and co-precipitates were: 2-4% CO ₂ , 35°C, 15-24hrs had the highest efficiency, (2) Circular DNA is more active than linear DNA, and (3) when the DNA concentration in the precipitation mixed solution is 20-30 μg/ml, the best precipitation can be formed (Chen C. and Okayama H., Mol. Cell. Biol., 1987, July, 2745). The method of (2) above is suitable for transient transfection. An earlier known method is to prepare a mixture of DEAE-dextran (Sigma#D-9885 MW5×10 ⁵ ) with a desired DNA concentration ratio, and perform transfection. Since most complexes are degraded in endosomes, chloroquine can be added to increase transfection efficiency (Calos, MP, Proc. Natl. Acad. Sci. USA, 1983, 80, 3015). The method of (3) above is called electroporation, and it is more widely used than methods (1) and (2) because it has no cell selectivity. Transfection efficiency can be maximized by optimizing the duration of pulsed current, pulse format, electric field strength (gap between electrodes, voltage), conductivity of buffer, DNA concentration, and cell density.

Among the above three methods, the method (2) using the transfection reagent is simple to operate and can detect a large number of samples with a large number of cells, so it is suitable for the present invention, preferably with Superfect TransfectionReagent (QIAGEN, #301305) and DOSPER Liposomal Transfection Reagent (Boehringer Mannheim #1811169).

Specifically, reconstruction from cDNA was performed as follows:

In plastic plates with 24-6 holes or 100mm Petri dishes, culture monkey kidney cells LLC-MK2 to 70-80% confluence with minimal essential medium (MEM), which contains 10% fetal bovine serum (FCS) and antibiotics (100 U/ml penicillin G and 100 μg/ml streptomycin). Recombinant vaccinia virus vTF7-3 expressing T7 polymerase was inactivated by exposure to UV for 20 minutes in the presence of 1 μg/ml psoralen, and then infected cells with 2 PFU/cell (Fuerst T.R. et al., Proc. Natl. Acad. Sci. USA, 1986, 83, 8122-8126; and Kato. A. et al., Genes Cells, 1996, 1, 569-579). The amount of psoralen added and the length of time exposed to UV can be adjusted appropriately. Cells are transfected after 1 hour of infection, and the method can be to use Superfect (QIAGEN company), 2-60 μ g (more preferably 3-5 μ g) above-mentioned recombinant Sendai virus cDNA, and the plasmid (24-0.5 μ g pGEM-N , 12-0.25 μg pGEM-P, and 24-0.5 μg pGEM-L, or more preferably 1 μg pGEM-N, 0.5 μg pGEM-P, and 1 μg pGEM-L) (Kato.A. et al., Genes Cells, 1996, 1 , 569-579) for lipofection, the plasmid expressing viral proteins can be used to generate the full-length Sendai virus genome and is required. Transfected cells were cultured in MEM without serum but containing 100 μg/ml rifampicin (Sigma) and cytarabine (AraC) (Sigma) at a preferred concentration of 40 μg/ml when needed so that The concentration of the drug is adjusted to be optimal so that the cytotoxic activity of the vaccinia virus is minimized and the recovery rate of the virus is maximized (Kato. A. et al., Genes Cells, 1996, 1, 569-579). Cells were cultured for 48-72 hr after transfection, then harvested and lysed by three freeze-thaw cycles. LLC-MK2 cells were transfected with cell lysates, cultured for 3 days to 7 days, and the medium was collected. In order to reconstruct a viral vector that lacks the gene encoding the envelope protein and cannot replicate, the vector can be used to transfect LLC-MK2 cells expressing the envelope protein, or transfected together with the expression plasmid of the envelope protein. Alternatively, the transfected cells are overlaid on the upper layer of LLC-MK2 cells expressing the envelope protein and cultured to propagate the defective viral vector (see PCT/JP00/03194 and PCT/JP00/03195). The titer of virus contained in the culture supernatant can be determined by measuring the hemagglutinin activity (HA). HA can be determined by the "internal point dilution method" (Kato. A. et al., Genes Cells, 1996, 1, 569-579). The resulting virus can be stored at -80°C.

There is no particular limitation on the type of host cells used for reconstitution, as long as the viral vector can be reconstituted in these cells. Host cells include cultured cells such as LLC-MK2 cells, monkey kidney-derived CV-1 cells, hamster kidney-derived BHK cells, and human-derived cells. Furthermore, in order to obtain a large amount of Sendai virus vectors, the vectors can be amplified by infecting fertile chicken eggs with the virus vectors obtained from the above-mentioned host cells. A method for preparing viral vectors using chicken eggs has been developed (Advanced protocols inneuroscience study III, Molecular physiology in neuroscience., Ed. by Nakanishi et al., Kouseisha, Osaka, 1993, 153-172). Specifically, for example, fertilized eggs are cultured in an incubator at 37-38° C. for 9-12 days to form embryos. The virus vector is inoculated into the allantoic cavity, and the fertilized eggs are further cultured for several days to propagate the vector. Conditions such as culture time can be changed depending on the type of recombinant Sendai virus used. Then, the allantoic fluid containing the virus is recovered. Sendai virus vectors can be isolated and purified from allantoic fluid samples by conventional methods (Tashiro M., Protocols in virus experiments., edited by Nagai and Ishihama, MEDICAL VIEW, 1995, 68-73).

When making defective viral vectors, for example, if two vectors with different envelope genes deleted in their genomes are transfected into the same cell, each missing envelope protein is provided by the expression of the other vector, and this complementation results in Infectious virus particles, which are capable of replicating and multiplying. That is, if two or more viral vectors of the present invention that are complementary to each other are mixed and inoculated at the same time, a large number of mixtures of various envelope gene-deficient viral vectors can be produced at low cost. Since these envelope gene-deficient viruses have smaller genomes, they allow the insertion of longer foreign genes. In addition, these non-infectious viruses are difficult to maintain a co-infection state after extracellular dilution, so they do not produce offspring and cause less damage to the environment.

If a virus vector is prepared using a disease-treating gene as a foreign gene, the vector can be administered for gene therapy. When the viral vector of the present invention is used for gene therapy, it can express the exogenous gene with desired therapeutic effect or the endogenous gene with insufficient supply in the patient's body by direct administration or indirect (ex vivo) administration. genes etc. There are no special restrictions on exogenous genes, including not only nucleic acids encoding proteins, but also nucleic acids that do not encode proteins, such as antisense nucleic acids or ribozymes.

The Paramyxovirus vector of the present invention can be combined with a desired pharmaceutically acceptable carrier to form a composition. Herein, "pharmaceutically acceptable carrier" refers to a substance that can be administered together with the carrier without inhibiting gene transfer through the carrier. For example, the paramyxovirus vector of the present invention can be appropriately diluted with physiological saline, phosphate buffered saline (PBS), etc. to prepare a composition. If the Paramyxovirus vector of the present invention is propagated in chicken eggs, the composition may contain allantoic fluid. The composition may also comprise a vehicle, such as deionized water or 5% dextrose in water. Stabilizers, antibiotics, etc. may also be included.

The paramyxovirus vector obtained by the above method or the composition containing the vector is introduced into skeletal muscle, so that the foreign gene carried by the paramyxovirus vector can be expressed in the skeletal muscle.

The administration of the viral vector may also be preceded by the administration of bupivacaine, which is known to enhance transgene expression by inducing muscle regeneration.

The type of gene introduced by the paramyxovirus of the present invention is not limited. Such genes may encode natural or artificial proteins. Natural proteins include hormones, cytokines, growth factors, receptors, enzymes, and peptides, among others. Such proteins may be secreted, transmembrane, cytoplasmic, and nuclear proteins, among others. Artificial proteins include fusion proteins (such as chimeric toxins), dominant-negative proteins (including soluble molecules of receptors and membrane-bound dominant-negative receptors), defective cell adhesion molecules, and cell surface molecules. Such proteins may also contain secretion signals, membrane localization signals, or nuclear translocation signals, among others. The introduced gene may be a gene not originally expressed in skeletal muscle. Alternatively, genes normally expressed in skeletal muscle can be introduced for overexpression. The function of unwanted genes expressed by skeletal muscle cells can also be inhibited by introducing antisense RNA molecules or ribozymes that cleave RNA.

It is expected that the vector of the present invention can be applied to gene therapy of various diseases. This type of gene therapy can compensate for expression defects in cells due to genetic defects, or confer new functions by introducing foreign genes into cells, or inhibit unwanted activity in cells by introducing genes that inhibit the activity of specific genes.

Diseases requiring gene therapy include muscular atrophy, myositis, myopathy, myopathy and cardiomyopathy after myocardial infarction, etc. Among them, dystrophin and IGF-I are effective genes in the treatment of muscular dystrophy, IGF-I and FGF in the treatment of myositis, and IL-10, IL-12, and IL-6 in the treatment of myopathy.

A preferable example of the gene introduced by the paramyxovirus of the present invention is the gene encoding insulin-like growth factor. The introduction of this gene is expected to significantly increase the number of regenerated muscle fibers and split muscle fibers that are indicators of hypertrophy, as well as increase the total number of muscle fibers. Therefore, the paramyxovirus introduced with the gene encoding insulin-like growth factor is expected to be applied to the treatment of neuromuscular disorders. Neuromuscular disorders include ruptured nerve fibers due to trauma, amyotrophic lateral sclerosis, and spinal muscular atrophy, among others.

Gene therapy is carried out by administering a composition containing a paramyxovirus vector intramuscularly or extramuscularly to express a foreign gene in skeletal muscle cells. In addition, the composition can also be administered ex vivo. The methods for introducing the carrier into skeletal muscle include the percutaneous introduction method and the skin-cut direct introduction method. When introducing the vector, care must be taken to ensure that the epimysium is not damaged.

In order to introduce an effective amount of vector into skeletal muscle cells, the skeletal muscle must be given a sufficient amount of paramyxovirus vector. The term "effective amount" refers to the amount that can ensure that the gene is introduced into skeletal muscle by the method of the present invention, resulting in the desired therapeutic or preventive effect (at least in part). The paramyxovirus vector of the present invention containing a desired gene is administered in an effective amount to induce phenotype changes in cells into which the vector has been introduced and/or its surroundings. Preferably, the Paramyxovirus vector of the present invention containing the desired gene is administered to skeletal muscle in an effective amount to introduce the gene into a significant number of cells in the skeletal muscle and induce phenotypic changes in these cells. The term "significant number of cells" means that at least about 0.1% of the target skeletal muscle cells at the site of administration have introduced the gene through the vector of the present invention, preferably about greater than or equal to 1%, more preferably about greater than or equal to 5%, more preferably about greater than or equal to 10%, most preferably about greater than or equal to 20%.

Whether the gene is successfully introduced into the cells can be tested by methods known to those skilled in the art. For example, the transcription product of a gene can be detected by methods such as Northern hybridization, reverse transcription-polymerase chain reaction (RT-PCR), or RNA protection assay. Detection by Northern hybridization or RT-PCR can also be performed in situ. The detection of translation products can be carried out as follows: Western blotting, immunoprecipitation, RIA, enzyme-linked immunosorbent assay (ELISA), pull-down test, etc. using antibodies. To facilitate the detection of successful gene transfer, the protein to be expressed can be tagged, or a reporter gene can be inserted to ensure its expression. Reporter genes include, but are not limited to, genes encoding β-galactosidase, chloramphenicol acetyltransferase (CAT), alkaline phosphatase, and green fluorescent protein (GFP), etc.

The dosage and route of administration of the carrier can vary depending on the disease, patient's body weight, age, sex, symptoms, purpose of administration, type of transgene, etc., and can be appropriately determined by those skilled in the art. Preferably, the carrier (containing a pharmaceutically acceptable carrier) is used in an amount of about 10 ⁵ pfu/ml to 10 ¹¹ pfu/ml, more preferably about 10 ⁷ pfu/ml to 10 ⁹ pfu/ml, most preferably about 1×10 ⁸ pfu/ml ~5×10 ⁸ pfu/ml.

The virus-containing compositions of the present invention can be administered to subjects including all mammals, including humans, monkeys, mice, rats, rabbits, sheep, cows, dogs, and the like.

Brief description of the drawings

Fig. 1 is a photograph and diagram of hIGF-I expression in vitro. (A) is a photo of the expression of hIGF-I in the culture supernatant of L6 cells infected with the virus. Under the conditions described in brackets, samples taken from the culture supernatant were analyzed by Western blotting (1 = no virus, 2 = LacZ/SeV (moi = 0.1, 3 days), 3 = IGF-I/SeV (moi = 0.1, 3 days), 4=IGF-I/SeV (moi=0.1, 4 days)). (B) Time- and moi-dependent expression coordinates of hIGF-I from L6 cells infected with hIGF-I/SeV. The culture supernatants of cells infected with different moi were taken at different time points, and the amount of hIGF-I was detected by ELISA kit.

Fig. 2 is a photograph showing that hIGF-I carried by SeV strengthens the myogenic differentiation and hypertrophy of L6 cells.

Control cells were cultured for 4 days in serum-free medium (differentiation medium). Cells infected with virus at an moi of 0.05 or 0.2 were cultured in serum-free medium for 4 days. After fixation, myotubes were treated with a monoclonal antibody against the myosin heavy chain embryonic subunit (MAb BF-45), and other cells were stained for nuclei. Virus-infected cells showed moderate myotube hypertrophy at moi = 0.05 and more pronounced myotube hypertrophy at moi = 0.2 compared to cells cultured in serum-only depleted conditions. Nuclear organization centrally assembled in muscle fibers is found in virus-infected cells.

Figure 3 is a photograph of the possibility of transducing SeV into skeletal muscle in vivo.

Mature rats were injected with 200 μl of recombinant SeV (5×10e7 pfu) containing a LacZ reporter gene (LacZ/SeV) into the tibialis anterior muscle of mature rats with or without pretreatment with myonecrotic agent (bupivacaine). The muscles of each group were cut and stained after the following days to study the activity of β-galactosidase: A and B: bupivacaine (7 days), C and D: bupivacaine (14 days), E and F: bupivacaine (30 days), G and H: no bupivacaine (7 days), I and J: no bupivacaine (14 days), K and L: no bupivacaine (30 days).

Figure 4 is a photograph of the expression of hIGF-I in tibialis anterior muscle 7 days after virus injection, obtained by Western blot analysis with anti-hIGF-I antibody. LacZ/SeV (2×10e8 pfu) or hIGF-I/SeV (2×10e8 pfu) 200 μl was injected into the anterior tibialis muscle. Seven days later, 100 μg of frozen muscle tissue was excised, and 300 μl of tissue extract was obtained therefrom. Proteins in 50 μl extract (equivalent to 16.7 μg tissue) were precipitated with cold acetone, followed by western blot analysis with anti-hIGF-I antibody (M: labeled protein, 1: untreated, 2: LacZ/SeV (#1 animal ), 3: LacZ/SeV (#2 animal), 4: hIGF-I/SeV (#3 animal), 5: hIGF-I/SeV (#4 animal)).

Fig. 5 is a photograph of expression of hIGF-I in tibialis anterior muscle 7 days after virus injection. Regeneration of the tibialis anterior muscle. LacZ/SeV (2×10e8 pfu) 200 μl was injected into the right anterior tibialis muscle, and 200 μl of hIGF-I/SeV (2×10e8 pfu) was injected into the left anterior tibialis muscle. Transverse sections of dissected muscles were analyzed with hematoxylin and eosin (HE) (a, d and g), with acid phosphatase for detection of macrophages (b, e and h) and with BF-45 (c, f and i) Processing. Black and white arrows indicate BF-45 positive cells and macrophages, respectively.

Fig. 6 is a histogram of expression of hIGF-I in tibialis anterior muscle 7 days after virus injection. Number of myofibers expressing embryonic MyHC. The total number of embryonic MyHC positive cells was counted (n=4), and the results were expressed as mean±SD (n=4). A single asterisk indicates p<0.01 for pairwise comparisons (Student's t-test).

Figure 7 is a photograph of the regeneration of the tibialis anterior muscle 14 days after virus injection. LacZ/SeV (2×10e8 pfu) 200 μl was injected into the right anterior tibialis muscle, and 200 μl of hIGF-I/SeV (2×10e8 pfu) was injected into the left anterior tibialis muscle. Transverse sections of dissected muscles were stained with hematoxylin and eosin (A: allantoic fluid only, B: IGF-I/SeV (left), C: LacZ/SeV (right)).

Figure 8 is a photograph of the regeneration of the tibialis anterior muscle 30 days after virus injection. Almost no inflammatory response was found in the sections. Muscles treated with IGF-I/SeV split due to hypertrophy (Fig. 8a and b). The location of the split is indicated by the arrow. Regenerated myofibers treated with IGF-I/SeV almost returned to normal (Fig. 8c).

Figure 9 is a photograph of the regeneration of the tibialis anterior muscle 30 days after virus injection. The effect of hIGF-I expression on the number of myofibers (open bars) and split fibers (closed bars). The total number of fibers of the anterior tibialis muscle treated with LacZ/SeV in each animal of the first group was taken as the control treated with hJGF-I/SeV (this value was set as 1) (results were expressed as relative values with the control value) (empty column). Plotted (±SD) with mean (n=4) of relative values. Single asterisks indicate p<0.03 for pairwise comparisons (Student's t-test). The total number of split fibers was counted, and the results were expressed as mean ± SD (n=4). Double asterisks indicate p<0.01 for pairwise comparisons (Student's t-test).

Fig. 10 is a comparison diagram 3 days after the gene was introduced into the mouse leg muscles. The titer was 4×10 ⁷ CIU/mouse (100 μl, administered twice).

Best Embodiment of the Invention

The present invention will be described in further detail below with reference to examples, but the present invention is not limited to these examples.

[Example 1] Construction of Sendai virus vector

On the basis of the DNA sequence whose accession number is X00173, using primers 5'-ATCC GAATTC GCAATGGGAAAAATCAGCAGTC-3' (SEQ ID NO: 1) and 5'-ATCC GAATTC CTACATCCTGTAGTTCTTGTTTCCTGC-3' (SEQ ID NO: 2), by PCR The human IGF-I open reading frame (Nature, 1983, 306, 609-611) was amplified from a human cDNA library (Gibco BRL, Rockville, MD). The resulting PCR product was cloned into the EcoRI site of pBluescribeII and then sequenced. The product with the correct sequence of the hIGF-I gene was used with primers 5'-ATCC GCGGCCGC CAAAGTTCAGCAATGGGAAAAATCAGCAGTCTTC-3' (SEQ ID NO: 3) and 5'-ATCC GCGGCCGC GATGAACTTTTCACCCTAAGTTTTTCTTACTACGGCTACATCCTGTAGTTCTTGTTTCCTGC-3' (SEQ ID NO: 4) re-amplification, and then, clone the amplified product into the NotI site of the parental pSeV18+b(+) constructed for the preparation of the correct SeV full-length antigenome (+) sense RNA of 15402 nucleotides , to obtain pIGF-I/SeV. The resulting pIGF-I/SeV was transformed into LLCMK2 cells infected with vaccinia virus vTF7-3 expressing T7 polymerase. The T7-controlled full-length recombinant IGF-I/SeV RNA genome was packaged with the N protein, P protein and L protein obtained by drying each of the co-transformed plasmids. Virus was recovered after 40 hours of incubation, and the transformed cells were injected into embryonated chicken eggs to amplify the recovered virus.

[Example 2] In vitro expression of hIGF-1

The results of the study showed that IGF-I was associated with rat cell lines (J.Biol.Chem., 1997, 272, 6653-6662 and J.Biol.Chem., 1996, 135, 431-440) and mouse cell lines (J Biol.Chem., 1989, 264, 13810-13817) are closely related to proliferation, differentiation and hypertrophy. The present inventors investigated whether recombinant human IGF-I could induce morphological changes in L6 cells (neonatal rat myoblast cell line) through SeV-mediated gene transfer. With the newly constructed recombinant SeV (named hIGF-I/SeV) carrying the human IGF-I (hIGF-I) gene and the SeV (LacZ/SeV) encoding β-galactosidase, the following facts were studied: (1) The expression of IGF-I in the supernatant (Western blot analysis), (2) the expression kinetics of IGF-I in the supernatant (ELISA detection) and (3) the morphological changes of L6 cells.

in vitro studies

L6 cells were cultured in DMEM medium containing 20% FCS and penicillin/streptomycin to inhibit their differentiation into myotube cells. Cells were cultured in serum-free DMEM or, alternatively, infected with virus in serum-free, cells were cultured under differentiating conditions. For immunoblotting, 100 μl of supernatant was concentrated to 10 μl with 2 times the volume of cold acetone, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 15-25% separating gel. Then transferred to a polyvinylidene fluoride membrane (DAIICHIPURE CHEMICALS, Tokyo). Primary antibody reactions were performed with anti-human IGF-I monoclonal antibody (Diagnostic Systems Laboratories, Webster, TX) and secondary reactions were performed with horseradish peroxidase-labeled antibodies. Complexes were visualized with enhanced chemiluminescence. An ELISA test was performed to measure the IGF-I level in the supernatant. The method was basically according to the manufacturer's suggestion (R&D systems, Minneapolis, MN).

The result is as follows:

(1) After L6 cells were infected with hIGF-I/SeV at a multiplicity of infection of 0.1 for 3 days and 4 days, a reaction band with a molecular weight of 12-13 kDa was detected in the cell culture supernatant (Fig. 1A, lanes 3 and 4). This protein was not found in the supernatant of cells infected with LacZ/SeV. This proved that hIGF-I was produced by recombinant SeV and then secreted into the culture supernatant.

(2) Figure 1B shows the expression level of IGF-I in the supernatant of L6 cells infected with the virus. When the multiplicity of infection was 0.05, the concentration of IGF-I in the supernatant increased to 203ng/ml after 48 hours of infection, and did not induce cytopathic effect (CPE), and increased to 435ng/ml after 96 hours of infection. When the multiplicity of infection was 0.5, 566ng/ml and 935ng/ml of IGF-I were secreted into the medium after 48 hours and 96 hours of infection, respectively. When the multiplicity of infection was 2.5, the detected IGF-I concentrations were 39 ng/ml and 463 ng/ml 12 hours and 24 hours after infection, respectively. IGF-I was not found in supernatants infected with LacZ/SeV. This demonstrates that the gene product is secreted into the culture supernatant by SeV-mediated gene transfer.

(3) It is known that L6 cells do not express IGF-I but express IGF-I receptors, so they are responsive to exogenous IGF-I, thereby inducing hypertrophy (J. Cell. Biol. 1996, 135, 431-448) . The ability of recombinant IGF-I to induce differentiation of L6 cells was examined. Cultivate L6 cells with medium containing 20% FCS to proliferate. When 80% of the cells are confluent, the gene is mediated by the virus, and then cultured in serum-free medium or serum-free medium containing a certain concentration of hIGF-I protein. In order to inhibit the adsorption of hormones to the cell surface, 500 μg/ml bovine serum albumin was added to all samples. After 4 days, obvious differences in cell morphology were observed when the multiplicity of infection of the virus was different (Fig. 2). The resulting myotubes were treated with an anti-embryonic subunit myosin H chain (MyHC) antibody (MAbBF45). Bound antibodies were visualized with Alexa Flour (registered trademark) 568 goat anti-mouse IgG (H+L) conjugate. Nuclei were visualized by staining with propidium iodide. At a multiplicity of infection of 0.05 (Fig. 2A and E) and 0.2 (Fig. 2B and F), cells cultured in medium containing hIGF-I were compared with cells cultured in serum-free medium without hIGF-I ( Comparing Figure 2D and H), its myotubes are larger, with multiple nuclei in the center of the muscle fibers, and myotube hypertrophy, that is, the size and width of their myotubes are increased (Figure 2A, B, E, and F). Moreover, the morphological changes induced by virus infection were consistent with those induced by IGF-I protein (Fig. 2C and G). The above results indicated that the recombinant hIGF-I derived from SeV has the same potency as the IGF-I protein, and the recombinant hIGF-I also has biological functions in vitro, and can induce myogenesis of neonatal rat myoblast L6 cells.

[Example 3] In vivo expression of LacZ reporter gene

Sprague-Dawley rats (male, 6 weeks old, body weight 160-180 g) were anesthetized by intraperitoneal injection (50 mg/kg) of sodium pentobarbital. The hair on the hind limbs was shaved, and after wiping with ethanol, the skin was incised 1.5 cm to expose the tibialis anterior muscle. 200 μl of LacZ/SeV (5×10e7 pfu) was administered intraperitoneally to the left anterior tibialis muscle for the LacZ reporter gene transfer experiment. The expression of the introduced gene was observed by X-gal staining. Three days before vehicle administration, 200 µl of 0.5% bupivacaine solution was administered to the tibialis anterior muscle.

The results showed that muscle tissue pretreated with bupivacaine showed high levels of X-gal staining (Figure 3A and B) compared with untreated muscle (Figure 3G and H) after one week of LacZ/SeV administration ). In bupivacaine-pretreated muscles, some X-gal-positive fibers were observed in small myofibers surrounding necrotic fibers, infiltrated neutrophils, and macrophages (Fig. 3B). Intramuscular injection of bupivacaine and/or virus injection of mature rat tibialis anterior muscle induces such small fibers and they are regarded as regenerative fibers. Vitadello et al. (Hum. Gene Ther., 1994, 5, 11-18) found that after 3 days of bupacaine treatment, there were mononuclear cells and small myotubes stained by anti-MyHC monoclonal antibody (BF-45), The activity of enzymes introduced into naked DNA was higher at 3 days after injury compared with 1 or 7 days after injury. Positive small fibers that can introduce LacZ gene by SeV are regarded as myofibers with cell division activity or immature myotubes during skeletal muscle maturation. Viral infection-induced muscle fiber necrosis was much less extensive in animals not treated with bupivacaine than in animals treated with both bupivacaine and virus. The number of positive fibers was also less than that in bupivacaine-treated myofibers, but X-gal-labeled myofibers were also found in non-bupivacaine-treated myofibers (Fig. 3H). The cells surrounding the transduced myofibers lacked central nuclei and were larger in size than the regenerated myofibers shown in Figure 3B. This means that these cells may be mature muscle fibers. Considering the above facts, the transduced muscle fibers may be mature muscle fibers. Thus, SeV was able to transduce the gene encoding the protein into mature rat muscle fibers. X-gal expression was also observed 2 weeks after virus administration. In bupivacaine-treated/untreated animals, the appearance of positive fibers was the same as in animals treated with the above agents for 7 days, but their numbers were reduced (Fig. 3C, D, I and J). Thirty days after virus injection, X-gal-labeled fibers were found only in bupivacaine-treated animals (Fig. 3E and F). In animals not treated with bupivacaine, X-gal-labeled fibers were not found in muscle fibers, but were observed in interstitial cells (Fig. 3K and L). From the above results, it can be seen that SeV can infect mature myotubes in addition to myoblasts undergoing cell division and immature myotubes after cell division.

[Example 4] In vivo introduction of IGF-I gene

hIGF-I is highly expressed in vitro. In vivo, the LacZ gene was introduced into regenerated and mature myofibers by SeV-mediated gene transfer. After confirming these, it will be studied whether hIGF-I introduced into rat skeletal muscle through SeV-mediated gene transfer can promote the growth of skeletal muscle, such as the increase in the number of muscle fibers and the hypertrophy of myotubes. First, the degree of hIGF-I expression in tibialis anterior muscle was determined by Western blotting. The anterior tibialis muscle of mature Sprague-Dawley rats was given 200 μl each of allantoic fluid, 2×10e8pfu (4 times higher than that in previous experiments) hIGF-I/SeV virus solution and 2×10e8pfu LacZ/SeV virus solution .

After 1, 2 and 3 weeks of dosing, the animals were euthanized with a lethal dose of sodium pentobarbital. The anterior tibia was dissected for histological staining and western blotting. Human IGF-I in rat muscle was analyzed by Western blot. 100 μg of muscle tissue was frozen in liquid nitrogen and ground with a mortar and pestle. Add 500ml of 1M glacial acetic acid to each tissue, mix well, then place on ice for 2 hours, centrifuge at 3000×g for 15 minutes, and recover the supernatant. 1 M fresh acetic acid was added to the precipitate after centrifugation for re-extraction. Combine the supernatant obtained twice, freeze at -70°C, freeze-dry, and suspend in 300 μl of 50 mM Tris hydrochloric acid buffer (pH 7.8). Concentrate the solution from 50 μl to 10 μl for western blotting.

For immunohistostaining of regenerated muscle fibers, samples of tibialis anterior muscle snap frozen in isopentane under liquid nitrogen were used. The resulting samples were cut into 10 μm thick sections and mounted on glass slides covered with lysine. The immunohistostaining method of the regenerated muscle fibers is as follows: after the first antibody treatment of the slices with the mouse anti-embryonic myosin H chain monoclonal antibody (BF-45), at room temperature, the slices were treated with the second antibody biotinylated anti-mouse IgG (1:100; Vector, Burlingame, CA) was incubated for 1 hour, and then incubated with streptavidin-biotin complex (diluted at 1:200, Vector, Burlingame, CA) for 1 hour. hours for color development. The streptavidin-biotin complex was visualized with 0.05M Tris-HCl buffer (pH 7.6) containing 0.05% 3,3-diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide, followed by eosin Sections were counterstained. Monoclonal antibody BF-45 was developed by Dr. D. Schiaffino (Hum. Gene Ther., 1994, 5, 11-18), Padova, Italy and purchased from American Type Culture Collection (Manassas, VA). To assess the regenerative effect induced by hIGF-I, the number of anti-BF-45 immunopositive fibers was examined 7 days after administration. Furthermore, in order to evaluate the hypertrophic effect of hIGF-I, the number of split fibers in muscle was counted 30 days after administration.

The results showed that, one week after administration, the tibialis anterior muscle sample treated with IGF-I/SeV showed a band with a molecular weight of about 8-9 kDa using anti-hIGF-I antibody. This value (Fig. 4, lanes 4 and 5) is inconsistent with that observed in the culture supernatant (Fig. 1A). The hIGF-I precursor observed in in vitro experiments is different from that in vivo because it is digested by proteases and becomes mature hIGF-I (Bio Science, Cytokine Proliferation Factor, Yangtusha pp.104- 105), with a reported molecular weight of 7.7 kDa.

The effect of hIGF-I in normal mature muscle was next investigated. The left anterior tibialis muscle of mature rats (6 weeks old) was given hIGF-I/SeV (2×10e8 pfu), and the right anterior tibialis muscle was given LacZ/SeV (2×10e8 pfu), as group 1; only in the left anterior tibialis muscle The second group was given hIGF-I/SeV (2×10e8 pfu) (n=12); the third group was given LacZ/SeV (2×10e8 pfu) (n=12) only in the left anterior tibialis muscle ; Two anterior tibialis muscles were given allantoic fluid (n=3) as the fourth group.

In group 1, 7 days after the administration of IGF/SeV and LacZ/SeV, a large amount of necrosis was found in the muscle fibers of the remaining slices at the administration site, resulting in fiber decomposition, perimysial edema, and many mononuclear phagocytic cells outside the cells. Infiltration of macrophages, lymphocytes and several neutrophils (Figure 5d and g). The remaining normal-sized myofibers in the necrotic area were invaded by a large number of acid phosphatase-positive macrophages (Fig. 5c and h). However, no injury was observed in the muscle administered with allantoic fluid alone, and very few macrophages were found (Fig. 5a and b). Based on the above considerations, the introduction of SeV causes damage to the infected muscle and induces its necrosis, thereby causing the infiltration of macrophages and lymphocytes, etc., which can remove the necrotic muscle. However, if necrotic tissue remains, regeneration is hindered, so phagocytosis is extremely important for muscle regeneration. It is known that muscle precursor cells or satellite cells are activated after phagocytosis, and then myoblasts start to proliferate (Experimental Medicine, Yodosha, 2000, 3, 444-448). Furthermore, it was reported (Hum. Gene Ther., 1994, 5, 11-18) that after bupivacaine treatment for 3 days, the regenerated muscle fibers were composed of anti-MyHC monoclonal antibody (BF-45) positive monocytes and small Comprising myotubes, these cells can be directed to regenerate muscle fibers. The infiltrating macrophage population was interspersed with very small myofibers with a central nucleus, and the immune response of these cells to BF-45 was studied. A large number of BF-45 positive regenerated myofibers were observed in the left tibialis anterior muscle administered with hIGF-I/SeV (Fig. 5f) compared with the right tibialis anterior muscle administered with LacZ/SeV (Fig. 5i). As shown in Figure 6, the average number of BF-45 immunopositive fibers in the animals of group 1 were: LacZ/SeV (right) was 446 (n=4), IGF-I (left) was 1,722 (n =4). BF-45 immunopositive myofibers were absent in animals treated with allantoic fluid (Group 4, Figure 5c).

Fourteen days after viral vector administration, the number of macrophages was drastically reduced in muscles treated with hIGF-I/SeV and LacZ/SeV (Fig. 7) (groups 1, 2, 3). Myofibers (with central nuclei) of the same size increased moderately in LacZ/SeV-treated muscles (Fig. 7C). In the muscles treated with IGF-I/SeV there was a dissemination of macrophages and very few residual necrotic fibers (data not shown), and myofibers of various sizes including small fibers were observed (Fig. 7B), suggesting a continuous of new fiber formation. Fibroblast infiltration and resulting fibrosis were not observed in hIGF-I/SeV-treated muscle and LacZ/SeV-treated muscle. Seven days after virus introduction, it was found that the space occupied by mesenchymal cells dominated by macrophages was almost completely replaced by regenerated myofibers (Fig. 7B and C).

Thirty days after administration of viral vectors, in group 1 animals, the size of the treated muscles basically returned to normal and uniform (Fig. Among the myofibers (data not shown), compared with LacZ/SeV-treated muscles, the total number of myofibers in hIGF-I/SeV-treated muscles increased by 17% (p<0.03, n=4) ( FIG. 9 ). In Groups 3 and 4, there was no statistically significant difference in the number of myofibers between the left and right muscles (data not shown), and hypertrophy was found to often result in an increased number of split fibers. In group 1 animals, the average number of split fibers in hIGF-I/SeV-treated muscle was 6.1 times that of LacZ/SeV-treated muscle ( FIG. 9 ). The above data suggest that this splitting phenomenon may cause an increase in the total number of myofibers in hIGF-I/SeV-treated muscles.

There are several reports indicating the effect of IGF-I on mature skeletal muscle. Adans and McCue (J.Appl.Physiol., 1998, 84 (5), 1716-1722) studied the local injection of IGF-I to the tibialis anterior muscle of normal rats In terms of the effect of protein, it was found that muscle weight, total protein and total DNA increased significantly in the muscles injected with IGF-I. However, no increase in the total number of muscle fibers was reported. Barton-Davis et al. (Proc.Natl.Acad.Sci.USA, 1998, 95, 15603-15607) introduced IGF-I gene into aged rats with AAV vector and found fiber regeneration and muscle hypertrophy, but the number of regenerated muscle fibers did not increase. The inventors found in the research that the overexpression of hIGF-I leads to a significant increase in the regeneration fibers, muscle hypertrophy fibers and total number of muscle fibers of normal mature muscles. It is generally believed that the observed formation of new myofibers is caused by the high level expression of IGF-I by SeV.

[Example 5] Gene expression of purified LacZ-SeV/dF and LacZ-SeV in mouse leg muscles up to comparison

A comparative study of gene expression in leg muscle administration using BALB/c mice injected intramuscularly with an additive-type Sendai virus vector (LacZ-SeV) derived from the allantoic fluid of fertile chicken eggs and purified LLC/MK2/F/ Ad cell-derived F-deficient LacZ Sendai virus vector (LacZ-SeV/dF). The dose was 4×10 ⁷ CIU/mouse.

Eighteen BALB/c male mice (Chaya-Russ Riba Co., Ltd., Japan) were used in the experiment. Mice were enrolled at the age of 6 weeks, domesticated and fed for 2 days, divided into 3 groups, 6 mice in each group, respectively: (1) untreated group, (2) treated with LacZ-SeV (4×10 ⁷ CIU/mouse) Group, (3) LacZ-SeV/dF (4×10 ⁷ CIU/mouse) treatment group. The mice were anesthetized with ether, the legs were fixed, and 100 μl of each injection point was injected with a 29 G syringe at a volume of 2×10 ⁸ CIU/ml on both sides of the anterior tibialis muscle of each mouse. Rub the muscles for about one minute after dosing.

Three days after the administration, the mice were anesthetized with ether, dissected, and the leg muscles on the administration (right) side were taken for LacZ detection.

The quantitative method of LacZ is as follows: take leg muscle (2.0ml small test tube) and freeze it with liquid nitrogen. Add 500 μl lysate (approximately 500 mg muscle) and homogenate (MultiPro) under ice cooling. After centrifugation (15000 rpm x 15 minutes), 10 μl of the supernatant was taken and placed in an assay tube. Then add 70 μl of reaction buffer A (Galacton-Plus 1 μl + reaction buffer 99 μl = 100 μl kit; Galacto-Light-Plus, TROPIX, #250065), and place in the dark at room temperature for 30-60 minutes. Add 100 μl accelerator and measure with a luminometer.

Comparing the gene expression (quantitative) in the leg muscles 3 days after the administration, the results showed that the gene expression levels of the LacZ-SeV/dF and LacZ-SeV treatment groups were significantly higher than those of the untreated group ( FIG. 10 ). The results of LacZ-SeV/dF treatment group and LacZ-SeV treatment group were slightly different, but the expression levels were roughly the same. This is generally believed to be due to the lesser impact of secondary infection within the muscle. In gene therapy for muscle diseases, the use of F-deficient and additive viruses is expected to be equally effective because they do not differ in gene expression.

Industrial Applicability

The invention utilizes paramyxovirus vectors to efficiently introduce foreign genes into skeletal muscle. Therefore, the present invention provides a basic technology for targeted skeletal muscle gene therapy, especially for the treatment of systemic diseases and neuromuscular disorders. In addition, the use of the paramyxovirus vector of the present invention to introduce the gene encoding insulin-like growth factor into skeletal muscle is expected to be useful in the treatment of muscle fiber atrophy, reduction and degeneration. This could potentially enhance existing therapies for neuromuscular disorders.

Sequence Listing

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Claims (14)

1. A method for introducing an exogenous gene into skeletal muscle, the method comprising giving a paramyxovirus vector inserted into the exogenous gene to skeletal muscle. 2. The method of claim 1, wherein the paramyxovirus is Sendai virus. 3. The method of claim 1 or 2, wherein the exogenous gene is a therapeutic gene. 4. The method of claim 1 or 2, wherein the foreign gene is a gene encoding insulin-like growth factor. 5. A paramyxovirus vector, which is inserted with a foreign gene and used for introducing the foreign gene into skeletal muscle. 6. The vector of claim 5, wherein the paramyxovirus is Sendai virus. 7. The vector of claim 5 or 6, wherein the foreign gene is a therapeutic gene. 8. The vector according to claim 5 or 6, wherein the foreign gene is a gene encoding an insulin-like growth factor. 9. A composition for introducing a foreign gene into skeletal muscle, the composition comprising a paramyxovirus vector inserted with a foreign gene. 10. The composition of claim 9, wherein the paramyxovirus is Sendai virus. 11. The composition of claim 9 or 10, wherein the foreign gene is a therapeutic gene. 12. The composition of claim 9 or 10, wherein the foreign gene is a gene encoding insulin-like growth factor. 13. The composition of claim 12 for increasing regenerating and/or splitting myofibers in mammals. 14. The composition of claim 12 for use in the treatment of neuromuscular disorders.

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